Packaging cell

ABSTRACT

A virus-producing cell sustaining the ability to produce viruses at high titer is successfully constructed by expressing the virus structural gene under the regulation of EF1α promoter. In this virus-producing cell, the virus structural gene is ligated to a selection marker gene via IRES and domains other than the protein coding domain are eliminated from the DNA encoding virus structural proteins. Thus, reduction of the titer due to cell passages can be prevented and emergence of wild type viruses caused by unfavorable recombination of the virus genome can be inhibited.

This application is a National Stage Entry of PCT/JP00/03557, filed Jun.1, 2000, which claims priority to Japan 11/154,364, filed Jun. 1, 1999,and Japan 2000-17831, filed Jan. 21, 2000.

TECHNICAL FIELD

The present invention relates to a virus-producing cell (packaging cell)that stably retains the ability to produce high titer viruses and theuse of such a cell for virus production.

BACKGROUND ART

Retroviral vectors are capable of transferring genes with highefficiency and stability to host cells. Thus, they are used in genetransfer methods, for transferring genes into cells in a variety offields including the field of medicine. Generation of retroviral vectorshas been developed based on the genetic structure and the life cyclemechanism of retroviruses. The fundamental principle is to transfer thegene of interest into a retroviral vector possessing a packaging signalbut lacking each of the gag, pol, and env structural genes, thenintroducing the retroviral vector into a packaging cell possessing eachof the gag, pol, and env structural genes lacking a packaging signal.Then, a virus particle containing the retroviral vector RNA is formed(i.e. packaged), and finally, retroviruses are produced into thesupernatant of a cell culture (Kitamura, T., International Journal ofHematology, 67:351–359 (1998)). Retroviruses produced in this manner canefficiently transfer the genes of interest into cells. Simultaneously,since they lack each of the gag, pol, and env structural genes, they canreplicate only within the packaging cells and cannot replicate withinnormal cells. Therefore, functional retroviruses are unlikely toregenerate from the prepared infected cells.

The production of packaging cells with such characteristics is disclosedin international publication numbers WO90/02806, WO94/19478, WO96/34098,and so on. However, the prior art is unsatisfying in terms of infectionefficiency and long-term stability. In addition, to increase the titerof the viruses, it is necessary to express the viral structural proteinsin a large amount within the packaging cell. Packaging cells that canexpress a large amount of viral structural proteins and show a limitedreduction in titer of viruses produced through passages are needed inthe art.

DISCLOSURE OF THE INVENTION

The object of this invention is to provide a virus-producing cell havingthe ability to produce infectious viruses at high titers. In a preferredembodiment of the present invention, virus-producing cells with enhancedlong-term stability and safety are provided. Another object of thisinvention is to provide a method for producing infectious viruses athigh titers using the virus-producing cells.

To accomplish the objects described above, the present inventors firstsearched for a promoter having a high activity in 293T cells. Uponinvestigating the strength of transcription activity of the SV40promoter, SRα promoter, EF1α promoter, TK promoter, MuLV LTR, and CMVLTR, the present inventors found that the activities driven by the EF1αpromoter and the CMV promoter were remarkably high compared to those ofthe other promoters. Accordingly, an attempt was made to establishhigh-performance packaging cells using the EF1α promoter.

Specifically, the present inventors used only coding sequence for thegag-pol and env genes in the packaging constructs under the regulationof the EF1α promoter in order to enhance the expression efficiency ofthe genes encoding the viral structural proteins in the packaging cells.Furthermore, to enhance the translation efficiency of the viralstructural proteins from the transcribed mRNAs, Kozak's consensussequence (GCCACC) was inserted upstream of the translation initiationcodon of these genes. In addition, a selective marker resistant gene waslinked via the IRES sequence (internal ribosomal entry site) downstreamof these genes, so that the genes positioned upstream and downstream ofthe IRES are translated from a single mRNA. This enabled reliableselection of cells expressing the inserted construct by the selectivemarker. In this manner, cells were produced having superior long-termstability compared to conventional packaging cells by separatelyintroducing a construct containing the selective marker resistant geneand, a construct containing the gag-pol and env genes.

In addition, improvement in safety was achieved by inserting the gag-poland env genes produced by PCR amplification of only the viral structuralproteins encoding regions into the construct, thus eliminating thepossibility of emergence of wild type viruses from the packaging cellsdue to recombination.

The present inventors examined the performance of the packaging cellsproduced in this manner and found out that the packaging cells producedhigh-titer retroviruses. Even after a long-term passage of 4 months,they retained the ability to produce retroviruses at the same level oftiter as before. The packaging cells of this invention are useful forproducing vectors for gene transfer to a living body, and may bepreferably utilized especially for producing high-titer retroviralvectors used for gene therapy, and such.

That is, the present invention relates to methods for producing avirus-producing cell having superior safety, retaining the ability toproduce infectious viruses at high titer even after long-term passages,and to methods for producing infectious viruses at high titer using thevirus producing cells. More specifically, this invention provides thefollowing:

-   (1) a cell for the production of retroviruses, wherein the cell has    an expression construct comprising a DNA encoding retroviral    structural proteins operably linked downstream of an EF1α promoter;-   (2) the cell according to (1), wherein the DNA encoding retroviral    structural proteins is a DNA encoding any one or more of the    proteins selected from the group consisting of gag, pol, and env;-   (3) the cell according to (2) that expresses all of gag, pol, and    env;-   (4) the cell according to (3), which has an expression construct    expressing gag and pol, and an expression construct expressing env;-   (5) the cell according to (3) or (4), wherein the env is derived    from either an ecotropic retrovirus or amphotropic retrovirus;-   (6) the cell according to any one of (1) to (5), wherein a Kozak's    consensus sequence is placed upstream of the translation initiation    codon of the DNA encoding the retroviral structural proteins in the    expression construct;-   (7) the cell according to any one of (1) to (6), wherein the DNA    encoding the retroviral structural proteins is bound via the IRES    sequence to a DNA encoding a selective marker;-   (8) the cell according to any one of (1) to (7), wherein the DNA    encoding the retroviral structural proteins is substantially free of    virus genome-derived DNA with the exception of the protein coding    region;-   (9) the cell according to any one of (1) to (8), wherein the cell is    derived from 293 cells;-   (10) the cell according to (9), wherein the cell is derived from    293T cells;-   (11) the cell specified by the accession No. FERM BP-6737 or FERM    BP-6977;-   (12) a method for producing a retrovirus, comprising the steps of:    introducing a retroviral vector DNA lacking at least one of the    genes encoding a viral structural protein into the cell of any one    of (1) to (11);-   (13) the method according to (12), wherein the retroviral vector DNA    lacks all of the genes encoding the gag, pol, and env;-   (14) the method according to (12) or (13), wherein a foreign gene is    included in the retroviral vector DNA; and-   (15) a retrovirus produced by the method of any one of (12) to (14).

The packaging cells of this invention are characterized by their use ofthe EF1α promoter for the expression of retroviral structural proteins.The present inventors searched for a promoter that has a high activityin packaging cells. As a result, it was found out that the EF1α promoterhad an especially strong activity among the promoters whose activitycould be detected. The packaging cells of this invention highly-expressthe retroviral structural protein due to the use of the EF1α promoter,which, in turn, enables the production of high-titer virus particles.

A DNA encoding the retroviral structural protein(s) is expressed underthe regulation of the EF1α promoter in a packaging cell by producing anexpression construct in which the DNA encoding the retroviral structuralprotein(s) is operably linked downstream of the EF1α promoter, and then,introducing it into the cell. The term “operably linked” used hereinindicates that the EF1α promoter is bound to the DNA such that itsactivation ensures the expression of the downstream DNA encoding theretroviral structural protein.

Examples of the retroviral structural proteins expressed in the cellsare the gag, pol, and env. Theoretically, it is not necessary to expressall of these proteins in the packaging cells and it is possible to placegenes encoding some of these proteins on the retroviral vector DNA. Forexample, it is possible to have the packaging cells express the gag andpol, whereas the env gene is placed on the retroviral vector DNA.However, in this case, there is the possibility that the amount of envexpression would not reach the required amount. Therefore, it ispreferable that all of gag, pol, and env are expressed by the packagingcells themselves.

The expression construct is preferably separated into a constructexpressing the gag and pol, and a construct expressing the env, whichare then introduced into the cell. In this manner, the possibility thatself-replicating viruses will be produced, due to the recombination ofpol, gag, and env that often occurs among those existing on theretroviral vector and those in the packaging cell, will be reduced. Thisis important from the view of safety, for example, when using theviruses produced by these cells in gene therapy. Regarding gag and pol,it is preferable to have them encoded as gag-pol on a same construct.This is because it is known that the expression ratio between pol andgag is important for the production of high-titer viruses and expressionof pol alone at large quantities, which may occur by making separateexpression constructs of gag and pol, will cause toxicity towards thecell.

It is possible to prepare packaging cells using env, if desired, derivedfrom ecotropic retroviruses (referred to as ecoenv) or amphotropicretroviruses (referred to as amphoenv). Packaging cells having ecoenvproduce ecotropic retroviruses whereas packaging cells having amphoenvproduce amphotropic retroviruses. Since the ecotropic retrovirus has aglycoprotein binding to the ecotropic receptor that exists only on thecell surface of rat and mouse, they only infect rat and mouse cells. Onthe other hand, the amphotropic retrovirus is capable of infectingvarious species, such as rat, mouse, humans, chicken, dog, cat, etc. Anexample of a retrovirus used in gene therapy is the amphoenv, which iscapable of infecting humans. However, to clone novel genes in thelaboratory, it is safer to use retroviruses produced from packagingcells that carry ecoenv with no infectivity to human.

Various retroviral env, such as env derived from Rous sarcoma virus(RSV), may be used (Landau, N. R. and Littman, D. R., (1992) J. Virology5110–5113)). Furthermore, envelope proteins other than those fromretroviruses may be used. For example, it is possible to use vesicularstomatitis virus (VSV)-derived G protein (VSV-G) (Ory, D. S. et al.,(1996) Proc. Natl. Acad. Sci. USA 93: 11400–11406).

In a preferred embodiment of this invention, the Kozak's consensussequence (GCCACC) is placed upstream of the translation initiation codon(ATG) of the retroviral structural protein gene within the expressionconstruct to increase the translation efficiency of mRNA encoding theseproteins transcribed from the expression constructs for expression ofretroviral structural proteins (the Kozak's rule reveals that it ishighly probable that a GCCACC sequence exists in front of thetranslation initiation site ATG).

In another preferred embodiment, this invention provides packaging cellsharboring expression constructs that express the viral structuralprotein(s) and a selective marker simultaneously by operably linking theDNA encoding the viral structural protein(s) and selective marker viathe IRES. The viral structural protein(s) and selective marker areencoded on a single molecule transcribed by EF1α activation in theexpression construct. Thus, not only the protein (s) but also theselective marker is translated by the action of IRES from the RNAmolecule. Accordingly, reliable selection of cells expressing theretroviral structural protein(s) by the selective marker becomespossible due to the transformation with the expression construct.Conventionally, packaging cells were produced by separately introducingan expression construct carrying the selective marker resistant gene andan expression construct carrying the gag-pol and env genes into cells.Thus, the cells harboring the selective marker resistant gene and thoseharboring the viral structural protein genes were not always consistent,which caused problems in terms of the stability of the cells. Theutilization of the IRES sequence enables the preparation of packagingcells with excellent long-term stability.

In addition to blasticidin and puromycin described in the examples, forexample, hygromycin, diphtheria toxin, neomycin, and such may be used asselective markers. However, blasticidin and puromycin are preferablesince they act quickly and require shorter time for selection of cellsas compared to other drugs. The resistant genes of diphtheria toxin andhygromycin are described in “Bishai, W. R. et al., J. Bacteriol., 169:1554–1563 (1987)” and “hygromycin: Yin, D. X. et al., Cancer Res., 55:4922–4928 (1995)”, respectively.

In a further preferred embodiment, this invention provides cellsharboring an expression construct in which the DNA encoding retroviralstructural protein(s) under the regulation of EF1α is substantially freeof DNA other than that of the protein coding region. In the packagingcells of this invention, it is preferable to maximally remove viralgenome-derived sequences that are not essential for expression ofstructural proteins. This allows one to cut down to a minimum the riskof emergence of replication-capable retroviruses (RCR) by reducing thepossibility of recombination between the viral genome-derived DNA andretroviral DNA within the expression construct after the retroviralvector DNA is transferred into packaging cells that have above-mentionedexpression constructs. This, in turn, enables improvement of the safetyof the virus particles produced from the packaging cells.

DNA that is substantially free of DNA, other than that of suchretroviral structural protein coding regions, may be obtained, forexample, by a polymerase chain reaction using the viral genome DNA asthe template and primers corresponding to the viral structural proteincoding regions as described in the following examples. Herein, “issubstantially free of” DNA other than that of the coding region meansthat DNA other than that of the viral genome-derived protein codingregion is 30 or less, preferably 10 or less, more preferably 5 or less,and most preferably 0 bases.

For example, NIH3T3 (mouse fibroblast), 293 (human fetal kidney cells)(Graham, F. L., J. Gen. Virol., 36, 59–72 (1977)), and such may be usedas host cells for the production of packaging cells. However, theinvention is not limited thereto, so long as the cells have a hightransfection efficiency.

The calcium phosphate method, electroporation method, and generaltransfection methods with lipofectamine (GIBCO BRL), Fugene (BoehringerMannheim), and such may be used to introduce expression constructs intothe cell. Drug selection may be used as the selection method. Forexample, blasticidin, puromycin, hygromycin, diphtheria toxin, neomycin,and such may be used as the drug for drug selection, without limitationso long as the drug tolerance gene is known.

Preferably, the retroviral vector DNA is introduced into each clone ofthe obtained packaging cells following limiting dilution of the cells.Then, through measurement of the titer of the produced viruses, cellsproducing viruses with the highest titer are selected and cloned.

There is no particular limitation on the retroviral vector DNA insertedinto the packaging cells. When the packaging cells are derived fromcells expressing the SV40 large T antigen, as in the 293T cells, the useof vector DNA bound to the replication initiation site of SV40 enablesan increase in the number of its copies produced within the packagingcells, and thus an increase in titer can be expected.

Introduction of retroviral vector DNA into cells can be carried out bythe same method as that described above for the introduction of theviral structural protein expression constructs into cells. Theretroviral vector of interest can be prepared by harvesting theretroviral particles released into the supernatant of the packaging cellculture after the introduction of the retroviral vector.

The retroviral vectors produced by the packaging cells of this inventionmay be utilized in comprehensive fields of research and medicine. Forexample, they may be used as vectors for expressing the gene of interestex vivo or in vivo in gene therapy and in the production of animalmodels. They may be also useful as vaccines, for expressing antigenicproteins and proteins that elevate immunological functions. Further,they may be also useful as in vitro gene transfer vectors for analyzinggene functions. Further, the vectors can be also used to produceproteins of interest. They are also useful as vectors for production ofa library that expresses nonspecific cDNA molecule species, such as acDNA expression library.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be specifically described by way of examplesas follows. However, this invention should not be limited to theseexamples.

EXAMPLE 1 FACS-GAL Analysis

FACS-GAL analysis (Steven, N. F. et al., Cytometry, 12, 291–301, 1991)was performed in order to utilize the promoter exhibiting the highestactivity, by comparing promoter activities in 293 cells and 293T cells.According to the method, each of the promoters to which the lacZ genewas linked downstream were transfected into cells, and the expressiondistribution of lacZ within those cells was investigated.

293 cells and 293T cells were plated at 2×10⁶ cells per 6-cm tissueculture dish, 16 to 24 hours before transfection. 3 μg of plasmid,having lacZ linked downstream of each of the promoters, and 9 μl ofFugene (Boehringer Mannheim) were mixed into 200 μl fetal bovineserum-free DMEM medium, then were left standing for 5˜10 minutes.Thereafter, the plasmids were gently added to the 6-cm dishes platedwith either 293 cells or 293T cells. The cells were removed after 24hours, and then were suspended into 50 μl PBS, followed by incubation at37° C. for 5 minutes. Then, 50 μl FDG (fluoresceindi-b-D-galactopyranoside, Molecular Probe, Eugene, Oreg., catalog No.F1179: 2 mM in 98% distilled water) pre-warmed at 37° C. was added.After incubation at 37° C. for 1 minute, 1 ml PBS was added and thecells were placed on ice for 2 hours. After 2 hours, 20 μl PETG (phenylethyl-b-D-thiogalactoside, Sigma, catalog No. P4902) was added to stopthe reaction, and the expression distribution of lacZ within the cellwas investigated by FACS. As a result, the promoter activity of EF1α wasdetermined to be the highest in 293T cells. Compared to the retroviralpromoter, LTR, the promoter activity of EF1α was approximately 100-foldhigher, and compared to other promoters, the activity was tens of timeshigher.

EXAMPLE 2 Amplification of the Selective Marker Gene

To use blasticidin and puromycin as the selective markers, PCR reactionwas carried out under the following conditions using the resistant genesof each markers (bs^(r): Kamakura, T. et al., Agric. Biol. Chem., 51,3165–3168, 1987; puro^(r): Buchholz, F. et al., Nucleic Acids Res., 24,3118–3119, 1996) as the templates.

The reaction mixture consisted 10 ng template DNA, 5 μl 10× KOD buffer,5 μl 2 mM dNTP, 2.5 μl of each 10 μM primers (the base sequence of theprimers are shown below), 2 μl 25 mM MgCl₂, and 1 μl 2.5 U/ml KOD DNApolymerase (TOYOBO).

The primers used for the blasticidin resistant gene (bs^(r)) were5′-AAAACATTTAACATTTCTCAACAAG-3′ (SEQ ID NO: 1) and5′-ACGCGTCGACTTAATTTCGGGTATATTTGAGTG-3′ (SEQ ID NO: 2), and those forthe puromycin resistant gene (puro^(r)) were 5′-ACCGAGTACAAGCCCACG-3′(SEQ ID NO: 3) and 5′-ACGCAGATCTTCAGGCACCGGGCTTG-3′ (SEQ ID NO: 4). Thetemperature conditions were 94° C. for 30 seconds, 25 cycles of 94° C.for 30 seconds•54° C. for 30 seconds•72° C. for 2 minutes, and 72° C.for 10 minutes. After restriction enzyme treatment of the blasticidinresistant gene (bs^(r)) with SalI and the puromycin resistant gene(puro^(r)) with BglII, electrophoresis followed by extraction from thegel was performed. QiaexII (QIAGEN) was used for the extraction.

EXAMPLE 3 Preparation of pMX-IRES-EGFP

The IRES (internal ribosomal entry site) sequence is a sequence existingin the 5′-noncoding region of the viral mRNA, and is thought to form acharacteristic secondary structure. Protein translation of the host isinhibited due to the recognition of this sequence by the ribosome thatinitiates translation, which allows a predominant translation of theviral protein. To link the selective marker resistant gene behind theIRES, pMX-IRES-EGFP (Nosaka, T. et al., EMBO J., 18, 4754–4765, 1999)was digested with NcoI. After ethanol precipitation, blunt ends weremade via Klenow reaction. After phenol/chloroform treatment and ethanolprecipitation thereafter, restriction enzyme treatment with SalI toinsert bs^(r), or treatment with BglII to insert puro^(r) was carriedout followed by ligations of each construct. Thus, the selective markerresistant gene was positioned behind the IRES. IRES-bs^(r) fragment wascut out from pMX-IRES-bs^(r) by digesting with NotI (TAKARA) and SalI,while IRES-puro^(r) fragment was cut out from pMX-IRES-puro^(r) withNotI and BglII.

EXAMPLE 4 Amplification of gag-pol and Ecotropic env

PCR for gag-pol and ecotropic env were performed under the followingconditions.

The reaction mixture consisted 10 ng template DNA (Shinnick, et al.,Nature, 293, 543, 1981), 5 μl 1× LA Taq buffer, 8 μl 2 mM dNTP, 1 μl ofeach 10 μM primers (the nucleotide sequences are shown below), and 0.5μl 5 U/ml LA Taq (TAKARA).

The primers used for amplification of gag-pol were5′-CGAATTCGCCGCCACCATGGGCCAGACTGTTACCACTCCCTTAA-3′(SEQ ID NO: 5) and5′-TACGCCGGCGCTCTGAGCATCAGAAGAA-3′ (SEQ ID NO: 6), and those used foramplification of ecotropic env were5′-CGAATTCGCCGCCACCATGGCGCGTTCAACGCTCTCAAAA-3′ (SEQ ID NO: 7) and5′-TACGCCGGCGCTATGGCTCGTACTCTAT-3′ (SEQ ID NO: 8).

The temperature conditions for gag-pol were 98° C. for 2 minutes, 20cycles of 98° C. for 20 seconds•68° C. for 3 minutes, and 68° C. for 8minutes. Those for ecotropic env were 98° C. for 2 minutes, 30 cycles of98° C. for 20 seconds•68° C. for 2 minutes, and 68° C. for 7 minutes.

After electrophoresis of the PCR products, DNAs were extracted from thegel with QiaexII (QIAGEN). These DNAs were subcloned into TA vectorsusing the Original TA cloning kit (Invitrogen), and were digested withEcoRI and NotI (TAKARA).

EXAMPLE 5 Construction of gag-pol Expression Vector and Ecotropic envExpression Vector

To express either gag-pol-IRES-bs^(r) or env-IRES-puro^(r) under thecontrol of EF1α, the respective sequences were inserted into pCHO(Hirata, Y. et al., FEBS Letter, 356, 244–248 (1994); Okayama, Y. etal., Biochem. Biophys. Res. Commun., 838–45 (1996); pCHO is derived frompEF-BOS (Mizushima, S. and Nagata, S., Nucleic Acids Res., 18, 5332,(1990)) as described below.

[pCHO (gag-pol-IR S-bs^(r))]

After restriction enzyme treatment of pCHO with BamHI (TAKARA), bluntends were produced via Klenow reaction. Following ligation to the SalIlinker d(CGGTCGACCG) (Stratagene) (SEQ ID NO: 9), it was digested withEcoRI and SalI (TAKARA). The gag-pol and IRES-bs^(r) fragments producedin Examples 2 and 3 were inserted to produce pCHO (gag-pol-IRES-bs^(r)).

[pCHO(ecoenv-IRES-puro)]

After restriction enzyme treatment of pCHO with EcoRI and BamHI(TAKARA), ecotropic env and IRES-puro^(r) produced in Examples 2 and 3were inserted to produce pCHO (ecoenv-IRES-puro).

EXAMPLE 6 Construction of Amphotropic env Expression Vector

While ecotropic env can infect only those cells derived from the samespecies, amphotropic env can infect a variety of cells. Using thisamphotropic env, the env-IRES-puro^(r) expression vector was constructedas in Examples 4 and 5. The reaction mixture was contained 10 ng plasmidin which the amphotropic env gene (4070A) (Ott, D. et al., J. Virol. 64.757–766, 1990) was inserted, 5 μl 10× KOD buffer, 5 μl 2 mM dNTP, 2.5 μlof each 10 μM primers (the nucleotide sequences are shown below), 2 μl25 mM MgCl₂, and 1 μl 2.5 U/ml KOD DNA polymerase (TOYOBO).5′-CGAATTCGCCGCCACCATGGCGCGTTCAACGCTCTCAAAA-3′ (SEQ ID NO: 10) and5′-ATGCGGCCGCTCATGGCTCGTACTCTAT-3′ (SEQ ID NO: 11) were used as primers.The temperature conditions were 98° C. for 3 minutes, 25 cycles of 98°C. for 15 seconds•65° C. for 2 seconds•72° C. for 30 seconds, and 72° C.for 10 minutes.

pCHO (ecoenv-IRES-puro) produced in Example 5 was digested with EcoRIand NotI, and blunted via Klenow treatment. The amphotropic env abovewas then ligated to produce pCHO (amphoenv-IRES-puro).

EXAMPLE 7 Establishment of Packaging Cells

The 293T cells derived from human mesonephric cells, (DuBridge, R. B. etal., Mol. Cell. Biol., 7, 379–387. 1987) were transfected with theprepared construct. The 293T cells were plated at 2×10⁶ cells per 6-cmtissue culture dish, 16˜24 hours before transfection. 3 μg pCHO(gag-pol-IRES-bs^(r)) and 9 μl Fugene (Boehringer Mannheim) were mixedwith 200 μl fetal bovine serum-free DMEM media, and the mixture was leftstanding for 5 to 10 minutes. Then, the mixture was gently added to the6-cm dish plated with 293T cells. Cells were removed 48 hours later,plated onto a 10-cm dish, and were added to them DMEM media containing10% fetal bovine serum (8 μg/ml blasticidin).

Approximately 10 days later, each of pCHO (ecoenv-IRES-puro) and pCHO(amphoenv-IRES-puro) were transfected similarly, and then were culturedin a medium containing both of puromycin (0.8 μg/ml) and blasticidin (8μg/ml). The packaging cells of interest were established by obtainingsingle clones by limiting dilution at the time when proliferation of thecells had occurred.

The packaging cell in which the ecoenv expression vector was introducedwas named “Platinum-E cell (PLAT-E cell)”. The cell was deposited as“Pt-E” to a depositary institution as described below.

-   (a) Name and address of the depositary institution    -   Name: National Institute of Bioscience and Human-Technology,        Agency of Industrial Science and Technology    -   Address: 1-1-3 Higashi, Tsukuba-shi, Ibaraki, Japan (Postal code        number: 305-8566)-   (b) Date of deposition (Date of original deposition): May 31, 1999-   (c) Accession number: FERM BP-6737

Moreover, the packaging cell in which the amphoenv expression vector wasintroduced was named “Platinum-A cell (PLAT-A cell)”. The cell wasdeposited as “Plat-A” to a depositary institution as described below.

-   (a) Name and address of the depositary institution    -   Name: National Institute of Bioscience and Human-Technology,        Agency of Industrial Science and Technology    -   Address: 1-1-3 Higashi, Tsukuba-shi, Ibaraki, Japan (Postal code        number: 305-8566)-   (b) Date of deposition (Date of original deposition): Dec. 22, 1999-   (c) Accession number: FERM BP-6977

EXAMPLE 8 Production of Retroviruses

To investigate the infection efficiency of the viral solution obtainedfrom the packaging cells, an infection experiment was performed by thefollowing method. Packaging cells (ecoenv-introduced PLAT-E cells) wereplated at 2×10⁶ cells per 6-cm tissue culture dish, 16 to 24 hoursbefore transfection. 3 μg vector DNA (pMX-GFP: Onishi, M. et al., Exp.Hematol., 24, 324–329, 1996), which is a retroviral vector carryingMoLuLV as its basic framework and to which GFP is inserted, and 9 μlFugene were mixed with 200 μl serum-free DMEM media, and was leftstanding for 5 to 10 minutes. This was then added gently to the 6-cmdish plated with the packaging cells.

The supernatant (virus solution) was collected 48 hours later and wascentrifuged at 3,000 rpm for 5 minutes. To 500 μl of this solution, 5 μlof 1 mg/ml Polybrene (Sigma) and 1 μl of ×1000 IL3(R and D) were added,and was infected to 1×10⁵ BaF/3 cells for 5 hours. After 5 hours, 500 μlRPMI1640 media (containing IL3) were added. 24 hours later, takingadvantage of the property of GFP expressed in the infected cells, thatis, the property that it is excited by light at a wavelength of 395 nmand cause emission of light at a wavelength of 509 nm, infectionefficiency was measured as the proportion of cells emitting light (i.e.infectively expressing) using FACScan (fluorescein activated cellsorter: Becton-Dickinson). The infection efficiency towards BaF/3 cellsreached 95%, even without concentration of the virus solution.

Measurement of infection efficiencies of the virus solutions obtainedfrom PLAT-E (ecoenv-introduced PLAT-E cells) and BOSC23, thawedsimultaneously from liquid nitrogen, using BaF/3 cells revealed that theinfection efficiencies after 7 days from the beginning of the passagewas 90% or more for both packaging cells. On the other hand, after 2months of passage, whereas the infection efficiency decreased to 23%when the BOSC23 (Pear et al., Proc. Natl. Acad. Sci. USA, 90:8392–8396,1993) was used, infection efficiency as high as that after 7 days wasconfirmed to be maintained even after 2 months of passage, as well asafter 4 months of passage (after 4 months from the beginning of thepassage, infection efficiency of 70% or more was maintained towardsBaF/3 cells) when PLAT-E was used. That is, for approximately 4 monthsit was possible to produce retroviruses at titers of approximately1×107/ml.

When the infection efficiency of virus solution obtained from PLAT-Acells was measured using BaF/3 cells, a value of 30% was confirmed onthe 7th day from passage initiation (titer of approximately 1×10⁶/ml).

EXAMPLE 9 Examination on the Safety of PLAT-E

It was examined whether retroviruses, which have acquired the ability toreplicate due to recombination (RCR; replication competentretroviruses), appeared or not while the retrovirus vectors wereintroduced to the cells.

16 hours after 5×10⁴NIH3T3 cells were plated onto 6-cm tissue cultureplates, the cells were infected with viruses using 1 ml of virussolution, to which 10 μl of 1 mg/ml Polybrene was added, produced byintroducing pMX-neo into packaging cells. 3 ml 10% FCS DMEM was addedafter 4 hours and cultivation was continued until confluence wasreached. Following one passage, the system was cultured until 50%confluence was reached in DMEM supplemented with Polybrene at a finalconcentration of 2 μg/ml. Then, the medium was exchanged and cultivationwas continued for 2 to 3 days in 2 ml of DMEM. The supernatant waspassed through a 0.45 μm filter, infected newly to NIH3T3 cells, andselectively cultivated in DMEM containing G418 (neo). If retroviruseshaving the ability to replicate were produced, a G418 resistant colonyshould have appeared. However, such colonies were not detected.

EXAMPLE 10 Examination on the Stability of Plat-E Cells Compared toThose of Bosc23 Cells and Phoenix-E Cells

The present inventors compared Plat-E cells in its initial progress toBosc23 cells and Phoenix-E (Nolan Laboratory in the Department ofMolecular Pharmacology/the Department of Microbiology and Immunology inthe School of Medicine at Stanford University) cells in terms of itsability or inability to produce retroviruses at a high titer withlong-term stability by transient transfection. The cultivationconditions for the three packaging cell lines were as follows: Accordingto the manufacturer's instructions, the Bosc23 cells were proliferatedin DMEM containing GPT selective reagent (Specialty Media, Lavallette,N.J., USA) supplemented with 10% fetal bovine serum. Phoenix-E cellswere classified by FACS using the expression of CD8 as an index, werecultured for one week in DMEM containing hygromycin (300 μg/ml) anddiphtheria toxin (1 μg/ml) supplemented with 10% fetal bovine serum, andthen were transferred to DMEM supplemented with 10% bovine fetal serumwhich doesn't contain hygromycin and diphtheria toxin. Plat-E cells weremaintained all the time in DMEM containing blasticidin (10 μg/ml) andpuromycin (1 μg/ml) supplemented with 10% fetal bovine serum. Theinfection efficiency of retroviruses produced from Bosc23 diminishedwithin 3 months and that of retroviruses produced from Phoenix-E cellsdiminished similarly. On the other hand, retroviruses produced fromPlat-E retained an average titer of approximately 1×10⁷/ml to NIH3T3cells for at least 4 months under conditions of drug selective pressureand an infection efficiency of 75% or more (maximum of 99%) to BaF/3cells when they were transfected transiently.

To compare the expression level of Gag-pol and env mRNA in Plat-E,Bosc23, and Phoenix-E packaging cell lines, Northern blot analysis wasperformed using cells cultured for 3 weeks. The expression level ofGag-pol and env mRNA in Plat-E cells were 4-fold and 10-fold more,respectively, compared to other cell lines.

RT activity in the cell lysate was also analyzed. The RT activity inPlat-E cells was detected to be at least twice as high as that in Bosc23and Phoenix-E cells. Furthermore, the expression level of the envprotein evaluated by antibody staining using antibodies raised againstthe env gene product was considerably higher than that in Bosc23 andPhoenix-E cells.

Therefore, it was indicated that Plat-E cells could produce retrovirusesat high titer with long-term stability.

INDUSTRIAL APPLICABILITY

A virus-producing cell that sustains the ability to produce infectiousviruses at high titer even after long term passages is providedaccording to the present invention. Additionally, a method for producinginfectious viruses at high titers using the virus producing cells isalso provided. The use of retroviral packaging cells of this inventionenables the stable provision of retroviruses with high titers. Inaddition, by minimizing the viral genome to be included in the packagingcell, the inventors successfully lowered the possibility of theemergence of undesirable recombinant viruses, such as replicationcompetent retroviruses (RCR). Therefore, the retroviral packaging cellsof this invention serve as powerful tools for producing retroviralvectors in fields of biology and medical research, and are useful forproducing gene transfer vectors used in gene therapy.

1. A 293T cell useful for the production of retroviruses, wherein thecell contains an expression construct comprising DNA encoding gag, pol,and env retroviral structural proteins operably linked downstream of anEF1α promoter.
 2. A 293T cell useful for the production of retrovirusesby expressing retroviral structural proteins gag, pol and env, whereinthe cell comprises a first expression construct expressing gag and polfrom an EF1α promoter, and a second expression construct expressing envfrom an EF1α promoter.
 3. The cell according to claim 1, wherein the envis derived from either an ecotropic retrovirus or an amphotropicretrovirus.
 4. A cell specified by Accession No. FERM BP-6737 or FERMBP-6977 as deposited at the National Institute of Bioscience andHuman-Technology in Japan.
 5. The cell according to claim 1, wherein aKozak's consensus sequence is located upstream of a translationinitiation codon of the DNA encoding the retroviral structural proteinsin the expression construct.
 6. The cell according to claim 1, whereinthe DNA encoding the retroviral structural proteins is linked to a DNAencoding a selective marker via an IRES sequence.
 7. The cell accordingto claim 1, wherein the DNA encoding the retroviral structural proteinsis substantially free from virus genome-derived DNA other than the DNAencoding gag, pol, and env.
 8. A method for producing a retrovirus, themethod comprising the step of introducing into the cell of claim 1 aretroviral vector DNA that lacks sequence encoding gag, pol, and env. 9.The method according to claim 8, in which a foreign coding sequence isincluded in the retroviral vector DNA.
 10. The cell according to claim2, wherein the env is derived from either an ecotropic retrovirus or anamphotropic retrovirus.
 11. The cell according to claim 2, wherein aKozak's consensus sequence is located upstream of a translationinitiation codon of the DNA encoding the retroviral structural proteinsin each of the first and second expression constructs.
 12. The cellaccording to claim 2, wherein the DNA encoding the retroviral structuralproteins in each of the first and second expression constructs is linkedto a DNA encoding a selective marker via an IRES sequence.
 13. The cellaccording to claim 2, wherein the DNA encoding the retroviral structuralproteins in the first and second expression constructs is substantiallyfree from virus genome-derived DNA other than the DNA encoding gag, pol,and env.
 14. A method for producing a retrovirus, the method comprisingthe step of introducing into the cell of claim 2 a retroviral vector DNAthat lacks sequence encoding gag, pol, and env.
 15. The method accordingto claim 14, in which a foreign coding sequence is included in theretroviral vector DNA.
 16. A method for producing a retrovirus, themethod comprising the step of introducing into the cell of claim 4 aretroviral vector DNA that lacks sequence encoding gag, pol, and env.17. The method according to claim 16, in which a foreign coding sequenceis included in the retroviral vector DNA.